1. Field of the Invention
The present invention relates to a composition for detecting nitrites or nitrite-forming bacteria in foods and body fluids, particularly urine.
2. Description of the Prior Art
The frequency of occurrence of urinary tract infection is high. This disease is asymptomatic and the patients are women in most cases. When bacteriuria patients are left untreated, they suffer from manifest urinary tract infection frequently to cause various problems. However, an early diagnosis of the infection is possible by detecting bacteria excreted in the urine. When the diagnosis is to be effected biologically, urine is taken in such a manner that it is not contaminated with persistent bacteria. After a quantitative culture, at least 10.sup.5 /ml of the bacteria should be detected. However, the quantitative culture requires a complicated technique and a long time. Therefore, a chemical method has been employed for the rough quantitative determination of the bacteria more easily and rapidly.
Bacteria such as E. coli (the most common bacterium causing urinary tract infection), Proteus, Klebsiella, Staphylococcus and Enterococcus contain in their microbial bodies an enzyme which reduces nitrates into nitrites. Vesical urine of a healthy person is free from bacteria and, therefore, when a nitrite is detected in urine, it may be considered that a urinary tract such as renal pelvis or bladder is infected with one or more of the above-mentioned bacteria and that a nitrate in urine was reduced into a corresponding nitrite in the infected part.
A known, highly sensitive method of detecting bacteria in the urinary tract is based on the determination of the nitrite in excreted urine by a Griess test. A typical reagent for determining the nitrite by this test comprises an acidic solution of sulfanilic acid and 1-naphthylamine. In the Griess test, a nitrite contained in a sample diazotizes sulfanilic acid and a resulting diazonium salt is coupled with 1-naphthylamine to form a red dye. The concentration of this dye is proportional to the concentration of the nitrite. Therefore, the concentration of the nitrite can be determined by, for example, colorimetric analysis using a previously prepared calibration curve. This method had demerits in that the reagent solution is unstable and the operation is troublesome. To overcome these demerits, a method has been proposed wherein the Griess test is conducted on a carrier on which the reagent composition has been fixed.
In the field of so-called "dry chemistry" in which a reagent composition used for the assay of a body fluid composition is fixed on a carrier, a reagent composition comprising sulfanilic acid, 1-naphthylamine and a solid organic acid has been known (see Fischl & Pinto "Clin. Chim. Acta" 2, 527-533 (1957). Such a reagent mixture in dry state is far more stable than the solution so that it can be stored for a long time. Another example of the assaying agents is a reagent mixture comprising a diazotizable amine, an N,N-dialkyl-2-naphthylamine and a solid organic acid as disclosed in the specification of U.S. Pat. No. 3,415,717. However, these known test pieces have a minimum detection limit of 0.1 to 1 mg/100 ml in terms of sodium nitrite. Such a low sensitivity is insufficient for detecting only a trace of the nitrite to be detected in the examination of bacterial infection. Under these circumstances, the development of highly sensitive and stable test pieces has been demanded.
After intensive investigations made for the purpose of overcoming the above-mentioned defects of the conventional methods, the inventors have completed the present invention.